Differential Regulation of Apoptosis in Normal Versus Transformed Mammary Epithelium by Lutein and Retinoic Acid1

Vol. 9, 257–263, March 2000 Cancer Epidemiology, Biomarkers & Prevention 257

Differential Regulation of Apoptosis in Normal versus Transformed Mammary Epithelium by Lutein and Retinoic Acid1

Venil N. Sumantran, Rong Zhang, David S. Lee, and of considerable importance, because the therapeutic potential of Max S. Wicha2 these compounds depends on their selectivity in normal versus Department of Internal Medicine and Comprehensive Cancer Center, Cancer transformed cells. and Geriatrics Center, University of Michigan, Ann Arbor, Michigan Of the 600 known carotenoids, only 10% function as 48109-0942 vitamin A precursors in mammals (9). Therefore, understand- ing the role of nonprovitamin A carotenoids in cancer chemoprevention is important. Although ␤-carotene and the nonpro- Abstract vitamin A carotenoid canthaxanthin have been shown to inhibit We examined the effects of all-trans retinoic acid (ATRA) mammary carcinogenesis in mouse and rat models (1–4), the and lutein (a nonprovitamin A carotenoid), on apoptosis molecular mechanisms of their chemopreventive action remain and chemosensitivity in primary normal human unknown. Thus far, the suggested mechanisms of anticancer mammary epithelial cells, SV40 transformed mammary action of carotenoids include singlet oxygen quenching, immu- cells, and MCF-7 human mammary carcinoma cells. noenhancement, protection against cellular mutagenesis, and ATRA and lutein selectively induced apoptosis in up-regulation of specific connexins (gap junction proteins; transformed but not normal human mammary cells. In Refs. 10–12). Thus, there is growing evidence suggesting that addition, both compounds protected normal cells, but not the chemopreventive properties of carotenoids are independent transformed cells, from apoptosis induced by the of the antioxidant activity of these compounds (13). chemotherapy agents etoposide and cisplatin. Lutein is a nonprovitamin A carotenoid found in broccoli Furthermore, lutein and ATRA selectively increased the and spinach. Lutein has chemopreventive activity in mouse ratio of Bcl-xL:Bax protein expression in normal cells but models of murine breast and colon cancers (14, 15). It has been not transformed mammary cells, suggesting a possible suggested that lutein, as well as another carotenoid, zeaxanthin, mechanism for selective modulation of apoptosis. The can account for part of the decreased breast cancer risk for differential effects of lutein and ATRA on apoptotic women on high vegetable and fiber diets (2–4). Dietary lutein pathways in normal versus transformed mammary was associated with decreased breast cancer risk and estrogen epithelial cells may have important implications for receptor-positive status in premenopausal disease (16). chemoprevention and therapy. We examined the effects of both a retinoid, ATRA,3 and lutein on modulation of apoptotic pathways in normal human Introduction mammary epithelial cells as well as similar cells transformed with SV40. The latter cells have inactivated p53 and pRB There have been a number of investigations demonstrating the proteins (17). We also examined the effects of ATRA and lutein chemoprotective effects of selected retinoids and carotenoids. on MCF-7 cells. These cells are a fully transformed human This has been demonstrated in animal tumor models as well as mammary carcinoma cell line with wild-type p53 and high in epidemiological studies in humans (1–4). The molecular levels of Bcl-2 (18). In this study, we demonstrate the differ- mechanisms responsible for these protective effects are unclear. ential effects of these compounds on apoptosis and chemosen- However, one mechanism suggested for effects of retinoids on sitivity in normal versus transformed mammary cells. In addi- carcinogenesis involves the ability of these compounds to in- tion, we examined the effects of these compounds on duce apoptosis or programmed cell death in epithelial tumor expression of members of the Bcl-2 protein family. We report cells (5, 6). All-trans retinoic acid has been shown to induce that ATRA and lutein have differential effects on the apoptotic significant levels of apoptosis in breast, ovarian, and squamous threshold of normal versus transformed mammary cells and are carcinoma cells and can sensitize tumor cells to chemotherapy- associated with changes in expression of bcl-2 family members. induced apoptosis (5–8). Retinoids given at the high concen- These experiments have important implications for understand- trations necessary to achieve this effect, however, are toxic (5, ing the role of ATRA and lutein in chemoprevention and for 6). There have been fewer studies investigating the effects of developing strategies to increase the therapeutic index of cancer retinoids and carotenoids on normal breast epithelium. This is treatments.

Materials and Methods Received 6/21/99; revised 11/10/99; accepted 12/17/99. Reagents. ATRA and lutein were purchased from Sigma The costs of publication of this article were defrayed in part by the payment of Chemical Co. (St. Louis, MO) and Kemin Industries (Des page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by NIH Grants CA 61777-01 and CA 66233. 2 To whom requests for reprints should be addressed, at Department of Internal Medicine and Comprehensive Cancer Center, Room 6302, Cancer and Geriatrics 3 The abbreviations used are: ATRA, all-trans retinoic acid; THF, tetrahydrofu- Center, University of Michigan, Ann Arbor, MI 48109-0942. Phone: (734) 936- ran; MSU-1, Michigan State University-1 medium; SRF, serum replacement 1831; Fax: (734) 615-3947; E-mail: [email protected]. factor; PI, propidium iodide; CDDP, cisplatin.

Table 1 Effects of lutein and ATRA on apoptosis in normal versus transformed human mammary epithelial cells The normal cells, SV40-transformed cells, and MCF-7 tumor cells were grown in the presence of ATRA or lutein for 4 days. Apoptosis was quantitated after staining cells with PI. For each cell type, the values for the percentage of PI-positive cells should be compared with the corresponding value in the control (THF) sample. No significant increase in PI staining was observed in normal mammary cells treated with ATRA or lutein. In SV40-transformed cells, only ATRA (1.0 ␮M) significantly increased staining (n ϭ 2; P ϭ 0.028). In MCF-7 tumor cells, PI staining was significantly increased by ATRA (n ϭ 3; P ϭ 0.038 for both concentrations) and lutein (n ϭ 3; P ϭ 0.016) relative to that in the control (THF).

% of cells stained with PI (ϮSD) Normal mammary SV40-transformed MCF-7 breast cancer Sample cells cells cells THF 3.90 (1.00) 13.00 (2.00) 5.20 (1.10) 0.10 ␮M ATRA 4.80 (0.50) 26.50 (4.50) 16.10 (2.90) 1.0 ␮M ATRA 4.40 (0.30) 21.50 (0.50) 16.00 (3.00) Lutein 5.30 (1.00) 19.50 (7.50) 13.60 (1.05)

Fig. 1. Effects of ATRA and lutein on viability of normal and transformed mammary cells. Cell viability was measured on day 4 for each cell type. The graph shows the percentage of viable cells in ATRA- or lutein-treated samples Measurement of Apoptosis. Apoptosis was assessed by mon- with respect to the THF control for a given cell type. ATRA (1.0 ␮M) significantly decreased the viability of normal mammary cells with respect to the THF control itoring nuclear morphology after staining cells with PI, as (35% decrease; n ϭ 6; P ϭ 0.005). Both ATRA concentrations caused a signif- described (22). The Cell Death ELISA was performed as de- icant (30%) decrease in viability of MCF-7 tumor cells (n ϭ 6, P ϭ 0.001 for 0.10 scribed (23). ␮M; and n ϭ 3, P ϭ 0.004 for 1.0 ␮M). Lutein induced a significant decrease (17%) in viability of MCF-7 tumor cells (n ϭ 6, P ϭ 0.002). Ⅺ, normal cells; 2, Western Blotting. After 4 days of treatment with and without SV40-transformed cells; f, MCF-7 tumor cells. Bars, SD. THF, lutein, or ATRA, total lysates were prepared from attached and detached cells of each cell type. The Bradford assay was used to quantitate total protein within each sample. For normal cells, 20 ␮g of total protein of each sample were loaded Moines, IA), respectively. Each compound was dissolved in per lane. For tumor cells, 50 ␮g of total protein from each THF (99.9%; Aldrich Chemicals, Milwaukee, WI; Ref. 15), sample were loaded per lane. Selected blots were reprobed with which was stored under N2 gas and sealed to prevent peroxide an actin antibody to ensure equal loading of lanes. formation. Crystalline ATRA and lutein were stored in the dark Ϫ ␮ Western blot analysis was done as described (23), with at 20°C. Lutein was used at a final concentration of 7 M, antibodies to human Bcl-2, Bcl-xL, or Bax. The mouse mono- based on levels reported in sera of human subjects (19, 20). ␮ clonal Bcl-2 antibody and Rabbit polyclonal antibodies against ATRA was used at final concentrations of 0.10 and 1.0 M. human-Bcl-x were from Dako Corp. (Carpinteria, CA) and Stock solutions of ATRA and lutein (500-fold in THF) were Transduction Laboratories (Lexington, KY), respectively. The prepared for each experiment such that the final THF concen- mouse monoclonal antibody against human Bax and the goat tration in lutein/ATRA-treated samples was 0.20%. Control antimouse/antirabbit antibodies conjugated to horseradish per- samples were treated with THF alone (0.20%). oxidase were from PharMingen (San Diego, CA) and Ameresco Cell Culture. Normal human mammary epithelial cells were Corp. (Solon, OH), respectively. A chemiluminescence kit derived from reduction mammoplasties as described (21). Nor- (Amersham, Arlington Heights, IL) was used to visualize pro- mal mammary cells were cultured in MSU-1 medium ϩ 5% tein bands. In each experiment, three X-ray film exposures (10 fetal bovine serum (21). For experiments, cells were switched s to 2 min) of the same blot were scanned to calculate expres- to MSU-1 medium without serum but including SRFs. These sion levels of Bcl-xL, Bcl-2, or Bax proteins. factors include: human recombinant epidermal growth factor Ϫ8 Statistical Analysis. Significance of the effects of ATRA or (0.5 ng/ml), ␤-estradiol (10 M), insulin (5 ␮g/ml), hydrocor- Ϫ8 lutein in the three cell types were analyzed by the students tisone (0.50 ␮g/ml), thyroid hormone (T3 at 2 ϫ 10 M), and ϭ ␮ unpaired t test using a confidence level of 95% (P 0.05). human transferrin (5 g/ml). SV40 transformed cells are coun- Results of t tests are included in the figure legends. terparts of normal cells transfected with SV40 DNA and are grown in MSU-1-SRF medium ϩ cholera toxin (1 ng/ml; Sigma) and bovine pituitary extract (0.40%; Pel Freez, AR). RESULTS MCF-7 tumor cells were cultured in DMEM ϩ 5%, fetal bovine Effects of ATRA and Lutein on Growth and Apoptosis in serum, and 10 ␮g/ml insulin. Normal and Transformed Human Mammary Epithelial For experiments, the three cell types were seeded at 2 ϫ Cells. Studies have demonstrated that ATRA can induce ap- 104/well in 24-well plates, respectively, in MSU-1-SRF media. optosis in transformed mammary cells (5–7). To determine Each cell type was treated with THF, lutein, or ATRA for 4 whether this is also the case for the nonprovitamin A carote- days. Medium with and without compounds was replaced on noid, lutein, and to compare the effects of these compounds on day 2. Cell viability was measured on days 2 and 4 by trypan normal versus transformed cells, we investigated the effects of blue exclusion/Coulter counter. Both methods gave similar ATRA and lutein on growth and apoptosis in normal human results. For chemotherapy experiments, cells were pretreated mammary epithelial cells and similar cells transformed with with ATRA or lutein for 3 days, followed by treatment (24 h) SV40. We compared these effects to a fully transformed human with and without the drugs in the presence of fresh THF, lutein, mammary carcinoma cell line, MCF-7. or ATRA. ATRA reportedly binds serum proteins and has different

Table 2 Cell Death ELISA: Effects of lutein and ATRA on normal human mammary cells treated with/without CDDP and etoposide Normal human mammary cells were pretreated with and without lutein, ATRA, or THF for 3 days, followed by a 24-h exposure to CDDP or etoposide. Cyto- plasmic extracts from 3 ϫ 103 total cells (attached ϩ detached) per sample were isolated for the ELISA. The ELISA data represent the no. of units of cytoplasmic DNA-histone per sample. CDDP and etoposide each induced significant apoptosis in THF (n ϭ 2; P ϭ 0.043 for CDDP and P ϭ 0.050 for etoposide). In the presence of ATRA, neither drug caused significant apoptosis (n ϭ 2; P ϭ 0.273 for ATRA ϩ CDDP versus ATRA, and P ϭ 0.300 for ATRA ϩ etoposide versus ATRA alone). Lutein also blocked drug-induced death (n ϭ 2; P ϭ 0.375 for lutein ϩ CDDP versus lutein, and P ϭ 0.138 for lutein ϩ etoposide versus lutein alone).

Mean number of units of cytoplasmic DNA-histonea (ϮSD) Sample No drug CDDP Etoposide THF 1.00 (0.12) 3.50 (0.75) 4.40 (1.11) 0.10 ␮M ATRA 0.85 (0.17) 1.17 (0.25) 1.10 (0.19) Lutein 0.75 (0.15) 0.95 (0.20) 2.25 (0.87) a 1.0 unit DNA-histone at 405 nm ϭ the amount of horseradish peroxidase activity conjugated to anti-DNA-histone antibodies, specifically bound to a cytoplasmic extract from 3 ϫ 103 HL-60 cells treated with camptothecin (2 ␮g/ml for 4 h).

percentage of loss of viability in THF-, lutein-, or ATRA- treated samples is shown (Fig. 1). Lutein and ATRA (0.10 ␮M) did not significantly affect viability of normal or SV40-transformed mammary cells. However, ATRA (1.0 ␮M) significantly decreased viability of normal mammary cells. Fig. 1 also shows that ATRA (0.10 and 1.0 ␮M) and lutein significantly decreased the viability of MCF-7 tumor cells. To determine whether lutein and ATRA decreased viability in MCF-7 tumor cells by induction of apoptosis, we performed PI staining to quantitate apoptotic nuclei. Table 1 dem- onstrates the lack of induction of apoptosis by either ATRA or lutein in normal mammary epithelial cells. Thus, ATRA (1.0 ␮M) decreased viability (Fig. 1) without inducing apoptosis Fig. 2. A, ATRA and lutein protect normal mammary cells from chemotherapy- (Table 1) in normal cells. These data are consistent with a report induced cell death. Normal mammary cells were pretreated with THF, ATRA, or showing that ATRA induced apoptosis in transformed human lutein for 3 days. The cells were then treated for an additional 24 h with CDDP mammary cells but not in normal human mammary epithelial ␮ ␮ (50 M) or etoposide (100 g/ml) in the presence of fresh THF, ATRA, or lutein, cells (25). In SV40-transformed cells, only ATRA (1.0 ␮M) and the percentage of viability was determined. In the presence of the control solvent THF, etoposide and CDDP each induced significant cell death [(n ϭ 5, induced significant apoptosis. In contrast, both concentrations P ϭ 0.001) for each drug]. In the presence of lutein, etoposide and CDDP did not of ATRA induced a significant 3-fold increase in PI staining of induce significant death in normal cells (lutein ϩ etoposide versus lutein alone: MCF-7 tumor cells relative to the control (THF) sample. Lutein n ϭ 5, P ϭ 0.460) and (lutein ϩ CDDP versus lutein alone: n ϭ 5, P ϭ 0.500). also induced a significant (2.60-fold) increase in apoptosis in ATRA partially protected normal cells from apoptosis induced by etoposide (n ϭ 4, P ϭ 0.030 for ATRA ϩ etoposide versus ATRA alone) and CDDP (n ϭ 4, P ϭ MCF-7 tumor cells. The MCF-7 cell data represent an under- 0.002 for ATRA ϩ CDDP versus ATRA alone). Bars, SD. B, ATRA and lutein estimation of apoptosis induction because it was measured in do not alter chemosensitivity in MCF-7 breast cancer cells. MCF-7 tumor cells attached cells, and there were substantial numbers of detached were treated with THF, ATRA, or lutein in the presence or absence of etoposide cells, Ͼ90% of which were nonviable. The data in Table 1 and or CDDP as described in A. Each agent induced a significant increase in dead cell number in the presence of THF (n ϭ 3, P ϭ 0.005 for etoposide; and n ϭ 3, P ϭ Fig. 1 suggest that ATRA and lutein inhibit the viability of 0.003 for CDDP). In the absence of drugs, lutein alone caused a significant MCF-7 tumor cells by inducing apoptosis. However, neither increase in cell death (n ϭ 5, P ϭ 0.001). ATRA also induced significant cell compound induced apoptosis in normal mammary epithelial death in MCF-7 cells (n ϭ 5, P ϭ 0.017). Neither compound significantly cells. affected the sensitivity of MCF-7 tumor cells to CDDP or etoposide. Bars, SD. A and B: Ⅺ, THF; 2, 0.10 ␮M ATRA; f, lutein. Differential Effects of ATRA and Lutein on Chemotherapy- induced Apoptosis in Normal and Transformed Human Mammary Epithelial Cells. The above experiments demonstrated that both ATRA and lutein show selective induction of effects when used in vitro in serum-containing versus serum- apoptosis in transformed cells compared with normal human free media (24). Thus, all experiments on MCF-7 tumor cells mammary epithelial cells. Because a variety of chemotherapeu- were performed in the MSU-SRF used for culture of normal tic agents induce apoptosis, we examined the ability of ATRA cells and SV40 transformed cells, thereby facilitating compar- (0.10 ␮M) and lutein to alter the apoptotic threshold in normal isons between the three cell lines. However, similar results were and transformed human mammary epithelial cells. The higher obtained in serum-containing medium (data not shown). concentration of ATRA (1.0 ␮M) was not used because it The three cell lines were cultured in MSU-SRF medium, significantly inhibited the viability of normal mammary cells and viability was measured on day 4. For each cell type, the and MCF-7 tumor cells (Fig. 1).

Fig. 3. A, effects of ATRA and lutein and on Bcl-xL expression in normal and SV40-transformed cells. The graph shows Bcl-xL protein expression in normal and SV40-transformed cells after treatment with ATRA or lutein for 4 days. In normal cells, the fold changes in Bcl-xL expression induced by ATRA (n ϭ 3, P ϭ 0.025) are shown relative to Bcl-xL levels in the control (THF) sample. Lutein significantly induced Bcl-xL protein expression in normal cells (n ϭ 3, P ϭ 0.012). In SV40-transformed cells neither compound significantly affected Bcl-xL expression. Bars, SD. B, Bcl-xL expression assessed by Western blot. ATRA and lutein each induced Bcl-xL expression in normal mammary cells (top panel, Lanes 2 and 3) relative to the THF control (Lane 1). The bottom panel shows that Bcl-xL expression was not significantly affected by ATRA or lutein in SV40-transformed cells (Lanes 2 and 3 versus Lane 1).

Fig. 2A shows the effects of ATRA (0.10 ␮M) and lutein ratio of inhibitors of apoptosis such as Bcl-xL and Bcl-2 rela- on the viability of normal mammary cells treated with the tive to inducers of apoptosis such as Bax (26, 27). chemotherapeutic agents, etoposide or CDDP. In normal mam- To determine whether the effects of ATRA and lutein on mary cells, etoposide and CDDP induced a 3.0- and 4.50-fold apoptotic pathways were associated with modulation of the increase in dead cell numbers, respectively. However, the in- bcl-2 family of genes, we examined the effects of these com- duction of drug-induced apoptosis in normal mammary cells pounds on Bcl-2, Bcl-xL, and Bax expression. Normal, SV40 was completely blocked by lutein. Similarly, ATRA (0.10 ␮M) transformed, and MCF-7 carcinoma cells were exposed to decreased the sensitivity of normal cells to each drug by 2-fold. ATRA or lutein, and protein expression was assessed by West- Fig. 2B shows that MCF-7 tumor cells underwent signif- ern blotting. The average fold change in expression of each icant CDDP-and etoposide-induced cell death, which was not protein induced by a given compound was calculated relative to significantly affected by lutein/ATRA. In the absence of drug, its expression in the corresponding control (THF) sample. This ATRA and lutein each caused a significant (1.70- and 2.60- was done for each cell type, and the results were graphed in fold) increase in dead cell numbers, respectively, in MCF-7 Figs. 3–5. These figures also include representative Western tumor cells with respect to the control (THF). This result is blots showing the effects of lutein and ATRA on the expression consistent with the decreased viability and increased apoptosis of Bcl-xL, Bcl-2, and Bax in each of the three cell types. induced by ATRA and lutein in MCF-7 tumor cells (Fig. 1 and Fig. 3A shows that ATRA and lutein induced a 5- and Table 1). 7-fold increase, respectively, in Bcl-xL expression in normal To determine whether the chemoprotective effects of lu- mammary cells relative to the control (THF). Fig. 3B shows the tein and ATRA in normal cells were attributable to the modu- Western blot (Top panel, Lanes 2 and 3 versus Lane 1). In lation of drug-induced apoptosis, we performed the Cell Death contrast to normal mammary cells, ATRA and lutein did not ELISA, which measures DNA degradation in apoptotic cells significantly alter Bcl-xL expression in SV40 transformed cells (Table 2). After 24 h of exposure, both drugs induced signifi- (Fig. 3B, bottom panel). Data for the MCF-7 carcinoma cells cant apoptosis in normal mammary cells (3–5 fold). Table 2 are not shown because these cells (early passage) lack Bcl-xL shows that lutein and ATRA fully blocked CDDP and etopo- expression. side-induced apoptosis in normal cells. Fig. 2A also suggested Fig. 4 shows the effects of ATRA and lutein on Bcl-2 that lutein effectively protected normal cells from apoptosis expression in the three cell types. In normal cells, ATRA induced by both drugs. Thus, both by cell viability and the Cell induced a significant 3.50-fold increase in Bcl-2 expression Death ELISA assay, lutein and ATRA blocked chemotherapy- relative to the control (THF; Fig. 4A). Lutein did not induce a induced death in normal but not transformed cells. In MCF-7 statistically significant increase in Bcl-2 expression in normal tumor cells, the Cell Death ELISA showed that both drugs cells. The corresponding Western blot shows this data (Fig. 4B, induced a 2-fold increase in apoptosis, which was not signifi- top panel: Lanes 2 and 3 versus Lane 1). Neither compound cantly affected by ATRA or lutein (data not shown). This result significantly affected Bcl-2 expression in the SV40-trans- is consistent with the data shown in Fig. 2B. formed cells (Fig. 4B, middle panel) or in MCF-7 carcinoma Effects of ATRA and Lutein on Expression of Bcl-xL, Bcl-2, cells (Fig. 4, A and B, bottom panel). Similarly, expression of and Bax Proteins in Normal and Transformed Human the proapoptotic protein Bax was not significantly affected by Mammary Epithelial Cells. The experiments described above lutein or ATRA in any of the three cell types (Fig. 5). demonstrated that ATRA and lutein have differential effects on In summary, ATRA and lutein have differential effects on the induction and modulation of apoptosis in normal versus the expression of Bcl-2 family members in normal versus transformed human mammary epithelial cells, suggesting that transformed human mammary epithelial cells. Lutein selec- the apoptotic threshold may be differentially regulated by these tively increased the ratio of Bcl-xL:Bax in normal cells but not compounds in these cell types. The bcl-2 family of genes has transformed mammary cells. Similarly, ATRA selectively in- been demonstrated to play a key role in regulating the apoptotic creased the ratio of Bcl-xL ϩ Bcl-2:Bax in normal mammary threshold in many cell types. This threshold is modulated by the cells only. These data suggest a possible mechanism for the

Fig. 4. A, effects of ATRA and lutein on Bcl-2 expression in normal and transformed mammary cells. The graph shows the fold changes in Bcl-2 expression in normal, SV40-transformed cells, and MCF-7 tumor cells, after treatment with ATRA or lutein for 4 days. Bcl-2 expression was significantly induced by ATRA in normal cells (n ϭ 3, P ϭ 0.033) relative to the control (THF). In SV40-transformed cells and MCF-7 tumor cells, no significant changes in Bcl-2 expression were induced by ATRA. Lutein did not significantly alter Bcl-2 expression in any of the cell types. Bars, SD. B, Bcl-2 expression assessed by Western blot. The top panel shows that ATRA significantly increased Bcl-2 expression in normal mammary cells relative to the THF control (Lane 2 versus Lane 1). Bcl-2 expression was not significantly affected by ATRA in SV40-transformed cells (middle panel) or in MCF-7 tumor cells (bottom panel). Lutein did not significantly affect Bcl-2 expression in any of the cell types (Lane 3 versus Lane 1).

Fig. 5. A, effects of ATRA and lutein on Bax expression in normal cells, SV40-transformed cells, and MCF-7 cells. The graph shows fold changes in Bax expression in normal mammary cells, SV40-transformed cells, or MCF-7 tumor cells after treatment with ATRA or lutein for 4 days. Neither compound induced significant changes in Bax expression in any of the three cell types. Bars, SD. B, Bax expression assessed by West- ern blot. Lanes 2 and 3 show that Bax expression was unchanged by ATRA (Lane 2) or lutein (Lane 3) in normal cells (top panel), SV40 transformed cells (middle panel), and MCF-7 tumor cells (bottom panel), relative to their respective THF controls (Lane 1 of each panel).

ability of lutein and ATRA to protect normal cells, but not apoptotic threshold. Although the experiments were performed MCF-7 tumor cells, from apoptosis induced by etoposide and in serum-free medium to obviate the effects of serum-binding cisplatin. proteins, similar differential effects of ATRA and lutein on bcl-2 family expression and apoptosis were observed in normal Discussion mammary cells versus MCF-7 tumor cells in serum-containing medium (data not shown). Thus, the differential effects of In this study, we demonstrate that ATRA and lutein selectively induce apoptosis in transformed cells compared with normal ATRA and lutein on the apoptotic threshold of normal versus human mammary cells. The differential effects of these com- tumor mammary cells occur in the presence of serum and pounds on apoptosis and chemosensitivity in normal versus albumin that may be found in vivo. transformed mammary cells may in part be related to the The results demonstrating that lutein and ATRA raise the differential effects of these compounds on the expression of apoptotic threshold in normal mammary cells but induce ap- Bcl-xL, Bcl-2, and Bax expression in these cells. optosis in mammary carcinoma cells are consistent with reports ␤ This study is the first to demonstrate that lutein, a non- showing that the carotenoids -carotene and canthaxanthin are provitamin A carotenoid, has significant and markedly different selectively toxic in malignant tumor lines but not in normal effects on apoptosis in normal mammary cells compared with keratinocytes (28). Whether these and other carotenoids have transformed counterparts. Because lutein is a nonprovitamin A differential effects on apoptosis pathways in normal versus carotenoid, its mechanism of action is likely independent of the transformed cells remains to be determined. Interestingly, a retinoic acid receptor family. Whether lutein signals via a number of other chemopreventive agents, such as aspirin (29), specific receptor is as yet unknown. However, both compounds sulindac (30), and the retinoid N-4-hydroxyphenylretinamide selectively increased the ratio of Bcl-xL:Bax expression in (31), are thought to exert their action through selective induc- normal mammary cells and protected these cells from apoptosis tion of apoptosis in transformed cells. Our data suggest that the induced by etoposide and CDDP. Thus, these data suggest that chemopreventive effects of ATRA and lutein in mammary lutein and ATRA are similar in their ability to modulate the carcinogenesis may be attributable to similar mechanisms.

The molecular mechanisms responsible for the differential 6. Muindi, J. R. F. Retinoids in clinical cancer therapy. Cancer Treat. Res., 87: effects of ATRA and lutein on Bcl-2 family members in normal 305–342, 1996. versus transformed human mammary cells are unknown. How- 7. Liu, Y., Lee, M. O., Wang, H. G., Li, Y., and Zhang, X. K. Retinoic acid ␤ ever, elevated Bcl-2 expression has been observed in differen- receptor mediates the growth-inhibitory effects of retinoic acid by promoting apoptosis in human breast cancer cells. Mol. Cell. Biol., 16: 1138–1149, 1996. tiating hematopoietic, neural, and epithelial tissues (32). Inter- 8. Krupitza, G., Hulla, W., Harant, H., Grunt, T., and Dittrich, C. Retinoic acid estingly, ATRA-induced differentiation of neuroblastoma cells induced death of ovarian carcinoma cells correlates with c-myc stimulation. Int. was accompanied by marked induction of Bcl-2 and drug J. Cancer, 61: 649–657, 1995. resistance (33). We found that ATRA and lutein selectively 9. Goodwin, T. W. Metabolism, nutrition and function of carotenoids. Annu. increased the ratio of Bcl-xL:Bax expression in normal mam- Rev. Nutr., 6: 273–297, 1986. mary cells and protected these cells from apoptosis induced by 10. Rauscher, R., Edenharder, R., and Platt, K. L. In vitro antimutagenic and etoposide and CDDP. However, in MCF-7 tumor cells, ATRA anticlastogenic effects of carotenoids and solvent extracts from fruits and vege- and lutein induced significant apoptosis without altering the tables rich in carotenoids. Mutat. Res., 413: 129–142, 1998. ratio of Bcl-2:Bax expression, suggesting that these compounds 11. Zhang, L. X., Cooney, R. V., and Bertram, J. S. Carotenoids enhance gap can induce apoptosis, independent of regulation of expression junctional communications and inhibit lipid peroxidation in C3H/10T1/2 cells: relationship to their cancer chemopreventive action. Carcinogenesis (Lond.), 12: of bcl-2 family members (Table 1; Figs. 2B, 4, and 5). Candi- 2109–114, 1991. date genes known to be regulated by ATRA include p53, p21, ␤ 12. Rock, C. L., Kusluski, R. A., Galvez, M. M., and Ethier, S. P. Carotenoids c-myc, and transforming growth factor (34). Therefore, the induce morphological changes in human mammary epithelial cell cultures. Nutr. effects of ATRA and lutein on the expression of these genes in Cancer, 23: 319–333, 1995. normal versus transformed human mammary epithelial cells 13. Rock, C. L. Carotenoids. Biology and treatment. Pharmacol. Ther., 75: need to be investigated. 185–197, 1997. A variety of mammary carcinoma cells show increased 14. Chew, B. P., Wong, M. W., and Wong, T. S. Effects of lutein from marigold expression of either Bcl-2 or Bcl-xL compared with nontrans- extract on immunity and growth of mammary tumors in mice. Anticancer Res., formed mammary cells (35–37). We have suggested that the 16: 3689–3694, 1996. expression of these inhibitors of apoptosis may be required for 15. Kim, H., Araki, S., Kim, D. J., and Park, C. B. Chemopreventive effects of mammary transformation, because up to 80% of breast carci- carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhy- nomas overexpress one or the other of these gene products. drazine initiation. Carcinogenesis (Lond.), 19: 81–85, 1998. Thus, these genes may be constitutively expressed in trans- 16. Rock, C. L., Saxe, G. A., Ruffin, M. T., August, D. A., and Schottenfeld, D. Carotenoids, vitamin A, and estrogen receptor status in breast cancer. Nutr. formed mammary cells but regulated by retinoids and carote- Cancer, 25: 281–296, 1996. noids in nontransformed mammary cells. Although these stud- 17. Ludlow, J. W. Interactions between SV40 large-tumor antigen and the growth ies do not directly establish a causal relationship between suppressor proteins pRB and p53. FASEB J., 7: 866–871, 1993. modulation of Bcl-2 family members and apoptotic thresholds, 18. Monaghan, P., Robertson, D., Amos, T. A., Dyer, M. J., Mason, D. V., and other studies demonstrating chemoresistance by overexpression Greaves, M. F. Ultrastructural localization of Bcl-2 protein. Cytochemistry, 40: of Bcl-2 (or Bcl-xL) by transfection are consistent with this 1819–1825, 1992. mechanism (38, 39). 19. Prince, M. R., and Frisoli, J. K. Beta-carotene accumulation in serum and In summary, these studies demonstrate the selective ef- skin. Am. J. Clin. Nutr., 57: 175–181, 1993. fects of the nonprovitamin A carotenoid lutein and ATRA on 20. Potischman, N., McCulloch, C. E., and Byers, T. Breast cancer and dietary apoptosis pathways in normal versus transformed human mam- and plasma concentration of carotenoids and vitamin A. Am. J. Clin. Nutr., 52: mary epithelial cells. The chemopreventive properties of these 909–915, 1990. compounds may relate to their differential effects on apoptosis 21. Kao, C. Y., Nomata, K., Oakley, C. S., Welsch, C. W., and Chang, C. C. Two types of normal human breast epithelial cells derived from reduction mammo- pathways in normal versus transformed mammary cells. Fur- plasty: phenotypic characterization and response to SV40 transfection. Carcino- thermore, the selective protection of normal mammary cells genesis (Lond.)., 16: 531–538, 1995. compared with transformed cells by lutein suggests that clinical 22. Tewari, M., and Dixit, V. M. Fas- and tumor necrosis factor-induced apop- trials investigating the effect of this nontoxic compound on tosis is inhibited by the poxvirus crmA gene product. J. Biol. Chem., 270: chemotherapeutic efficacy and toxicity may be warranted. 3255–3259, 1995. 23. Sumantran, V. N., Ealovega, M. W., Nunez, G., Clarke, M. F., and Wicha, M. S. Overexpression of bcl-xS sensitizes MCF-7 cells to chemotherapy induced Acknowledgments apoptosis. Cancer Res., 55: 2507–2510, 1995. We gratefully acknowledge the gifts of SV40 transformed type II cells provided 24. Carpentier, Y., Mayer, P., Bobichon, H., and Desoize, B. Cofactors in in vitro by Dr. C. C. Chang from Michigan State University (MSU). We also thank Dr. induction of apoptosis by all-trans retinoic acid. Biochem. Pharmacol., 55: James Trosko (MSU) for helpful insight on the normal and SV40-transformed 177–184, 1998. human mammary cells. We thank Dr. Adam Drenowski for support for Rong Zhang. We appreciate Dr. Cheryl Rock’s help with project design and information 25. Seewaldt, V. L., Kim, J. H., Caldwell, L. E., Johnson, B. S., Swisshelm, K., on the properties of retinoids and carotenoids. Dr. David Beidler helped us design and Collins, S. J. All-trans retinoic acid mediates G1 arrest but not apoptosis of the chemotherapy experiments. normal human mammary epithelial cells. Cell Growth Differ., 8: 631–641, 1997. 26. Reed, J. C. Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance. Curr. Opin. Oncol., 7: 541–546, 1995. References 27. Yang, E., and Korsemeyer, S. J. Molecular thanatopsis: a discourse on the 1. Gerster, H. Anticarcinogenic effect of common carotenoids. Int. J. Vitam. Bcl-2 family and cell death. Blood, 88: 386–401, 1996. Nutr. Res., 63: 93–121, 1993. 28. Schwartz, J., and Sklar, G. Selective cytotoxic effect of carotenoids and 2. Krinsky, N. I. Effects of carotenoids in cellular and animal systems. Am. J. ␣-tocopherol on human cancer cell lines in vitro. J. Oral Maxillofac. Surg., 50: Nutr., 53: 2385–2465, 1991. 367–373, 1992. 3. Freudenheim, J. L., Marshall, J. R., Vena, J. E, and Laughlin, R. Premeno- 29. Elder, D. J., Hague, A., Hicks, D. J., and Paraskeva, C. Differential growth pausal breast cancer risk and intake of fruits, vegetables and related nutrients. inhibition by the aspirin metabolite salicylate in human colorectal tumor cell J. Natl. Cancer Inst., 88: 340–348, 1996. lines: enhanced apoptosis in carcinoma and in vitro transformed adenoma relative 4. Van Poppel, G. Carotenoids and cancer: an update with emphasis on human to adenoma cell lines. Cancer Res., 56: 2273–2276, 1996. intervention studies. Eur. J. Cancer, 29A: 1335–1344, 1993. 30. Shiff, S. J., Qiao, L., and Tsai, L. Sulindac sulfide, an aspirin-like compound 5. Lotan, R. Retinoids and apoptosis: implications for cancer chemoprevention inhibits proliferation causes cell cycle quiescence and induces apoptosis in HT-29 and therapy. J. Natl. Cancer Inst., 87: 1655–1657, 1995. colon adenocarcinoma cells J. Clin. Investig., 96: 491–503, 1995.

31. Oridate, N., Lotan, D., Mitchell, M. F., Hong, W. K., and Lotan, R. Inhibition 36. Naik, P., Karrim, J., and Hanahan, D. The rise and fall of apoptosis during of proliferation and induction of apoptosis in cervical carcinoma cells by reti- multi-stage tumorigenesis: down-modulation contributes to tumor progression noids: implications for chemoprevention. J. Cell. Biochem. Suppl., 23: 80–86, from angiogenic progenitors. Genes Dev., 10: 2105–2116, 1996. 1995. 37. Olopade, O. I., Adeyanju, M. O., Safa, A. R., Hagos, F., Mick, R., Thompson, 32. Zhang, K. Z., Westberg, J. A., Holtta, E., and Andersson, L. Bcl-2 regulates C. B., and Recant, W. M. Overexpression of Bcl-x in primary breast cancer is neural differentiation. Proc. Natl. Acad. Sci. USA, 93: 4504–4508, 1996. associated with high tumor grade and metastases. Cancer J. Sci. Am., 3: 230–237, 33. Lasorella, I., Iavarone, A., and Israel, M. Differentiation of neuroblastoma 1997. enhances Bcl-2 expression and induces alterations of apoptosis and drug resist- 38. Miyashita, T., and Reed, J. C. Bcl-2 gene transfer increases relative resistance ance. Cancer Res., 55: 4711–4716, 1995. of S49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation 34. Gudas, L. J. Retinoids, retinoid-responsive genes, cell differentiation, and induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res., cancer. Cell Growth Differ., 3: 655–662, 1992. 52: 5407–5411, 1995. 35. Silvestrini, S., Veneroni, S., Daidone, M. G., Benini, E., and Borscchi, P. The 39. Minn, A. J., Rudin, C. M., Boise, L. H., and Thompson, C. B. Expression of Bcl-2 protein: a prognostic indicator strongly related to p53 protein in lymph Bcl-xL can confer a multidrug resistance phenotype. Blood, 86: 1903–1910, node-negative breast cancer in patients. J. Natl. Cancer Inst., 86: 499–504, 1994. 1995.

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Venil N. Sumantran, Rong Zhang, David S. Lee, et al.

Cancer Epidemiol Biomarkers Prev 2000;9:257-263.

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